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a Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205-2179
b The Cell Structure and Function Laboratory, The Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231-1000
There is considerable evidence that mouse fertilization requires the binding of sperm to two of the three glycoproteins that form the zona pellucida (ZP), ZP3 and ZP2. Despite the biologic importance of this binding, no one has demonstrated that sperm express separate, saturable, and specific binding sites for ZP3 and for ZP2. Such a demonstration is a prerequisite for defining the distribution, numbers, affinities, and regulation of function of ZP3 and ZP2 binding sites on sperm. The experiments reported herein used fluorochrome-labeled ZP3 and ZP2 and quantitative image analysis to characterize the saturable binding of ZP3 and ZP2 to distinct sites on living, capacitated, acrosome-intact mouse sperm. Approximately 20% of the ZP3 binding sites were found over the acrosomal cap, and the remaining sites were located over the postacrosomal region of the head. In contrast, ZP2 binding sites were detected only over the postacrosomal region. Saturation analysis estimated numbers and affinities of the binding sites for ZP3 (Bmax 
185 000 sites per sperm; Kd 67 nM) and ZP2 (Bmax 500 000 sites per sperm; Kd 200 nM). Use of unlabeled ZP3, ZP2, and ZP1 as competitive inhibitors of the binding of fluorochrome-labeled ZP3 and ZP2 demonstrated that ZP3 and ZP2 bound specifically to their respective sites on sperm. Finally, we demonstrate that extracellular calcium as well as capacitation and maturation of sperm are required for these sites to bind their respective ligands.
1 This work was supported by grant 1 R01 HD-35699 from NICHD; by the Hopkins Population Center (P30-HD-06268); and by an institutional training grant from the National Institutes of Health (5T32 AG 00250) to C.L.K.
2 Correspondence: William W. Wright, The Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room 3508, 615 N. Wolfe St., Baltimore, MD 21205-2179. FAX: 410 614 2356; bwright{at}jhmi.edu
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